Caffeine is the most widely consumed psychoactive substance. It is estimated that caffeine consumption from all sources averages approximately 70 mg/person/day worldwide and -220 mg/day/person in the US and Canada. Moderate consumption of caffeine produces overall psychostimulant effects (reducing fatigue, enhancing performance) in humans with little risk of harmful side effects. Caffeine is known to act at a multitude of molecular targets. Therefore, depending on dose, caffeine can produce many different effects in the intact organism. Presently, caffeine is believed to exert its effects primarily by blocking brain adenosine, particularly AI and A2A receptors. However, the evidence is circumstantial and the possible contribution of other molecular targets, particularly in the untoward effects of higher doses, remains to be determined. The overall goal of the proposed studies is to conclusively assess the contribution of the AI and A2A receptors and of the central versus peripheral Aj/AaA receptors to caffeine's psychostimulant and cardiovascular effects in mature and developing animals. This proposal is built on our successful development of two novel adenosine receptor knockout models: the congenic Ai-A2A receptor double knockout and a conditional, tissue-specific A2A receptor knockout mouse. With these novel knockout models coupled with molecular, neurochemical and pharmacological analyses, we will test the hypotheses that: (1) forebrain A2A receptors are essential for the psychostimulant properties of caffeine, and combined blockade of AI and A2A receptors mediate most effects of caffeine in adult animals;(2) both AI and A2A receptors contribute to the cardiovascular response to acute caffeine, and (3) the short and long-term effects of caffeine on immature animals are distinct from effects in adults and are mediated by different molecular targets. The information derived from these studies will provide the clearest assessment yet of the role of subtypes of adenosine receptors in mediating caffeine's psychostimulant effect. This will also shed light on the long-term effects of perinatal caffeine exposure, which may have significant public health relevance. This knowledge will significantly enhance our understanding of caffeine's psychostimulant action, and provide a neurobiological basis for guidelines for healthy usage of caffeine as a stimulant to improve human performance.